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L0301P48 - Gene Therapy and Therapeutic Cloning
Cell Differentiation and Totipotency *differentiation (specialisation) results from differential gene expression *fertilised egg is totipotent **can give rise to all cell types of organism *as cells divide, become determined, and then differentiate, they generally lose totipotency —> become pluripotent *differentiation does not generally involve permanent changes in the genome *nuclear transplant and cloning experiments show that the differentiated cell nucleus can retain totipotency and have the ability to act like a zygote nucleus and stimulate the production of an entire organism Reverse Differentiation In Plants: *easier to reverse than in animal cells *root cells can be “tricked” into forming a new plant, which is genetically identical to the original plant (clone) In Animals - Frogs (first experiment): *nuclei obtained from tadpoles in various stages of development resulted in the formation of normal early embryos *nuclei of differentiated epithelial cells from the gut of feeding tadpoles produced fertile adult frogs *no success in making an adult frog by transplantation of an adult cell nucleus to an egg Animal Cloning Dolly the Sheep *egg from mother (Scottish Blackface sheep) was enucleated by a micropipette *enucleated egg fused with a donor nucleus from a Finn Dorset sheep *egg allows to develop into an embryo and via IVF Dolly was created **looked like a Finn Dorset sheep *showed that a cell taken from an adult organism can be given totipotency and differentiate into all types of cells of that organism Applications *pharming - animals producing protein in milk *cloned pigs - organs for human transplant patients (contain knockout gene which prevents human rejection of pig tissue) Stem Cells *are unspecialised cells *have the ability to proliferate and differentiate into other cell types Embryonic Stem Cells *from the inner cell mass of the early embryo *are pluripotent *with suitable environmental stimulation, can be cultured in the laboratory and induced to form cells that differentiate *e.g. treatment with a derivative of vitamin A causes ESC to form nerve cells Customisation of ESCs *transfer nuclei from a normal patient cell to an enucleated egg (somatic cell nuclear transfer SCNT) *allow it to develop and collect the ESCs from the embryo *collected cells could potentially provide immunologically matched cell grafts for the patient Legality *SCNT has been approved in Australia *Not permitted: **embryo development beyond 14 days **embryo implantation into a woman or animal body **production of embryos by egg and sperm for research Controversial Issues *not feasible for clinical treatment, **NT is relatively inefficient **limited availability of eggs **not perfect match of DNA ***mitochondrial DNA is from egg donor *most of the ESC lines are ‘contaminated’ through growth on animal products *need to determine the precise identity and properties of the derived specialised cells *non-specialised ESC can create teratomas (inappropriate development of mixed cell types which can lead to tumours) *essential to purify and use cells at a more specialised stage *ethical concern that SCNT requires the destruction of human embryos Adult Stem Cells *found in tissues such as brain, bone marrow, skin, intestine being responsible for their replenishment *however are rare and distinguishable by behaviour but not by appearance *not easy to grow and have a limited lifespan *already used for bone marrow transplants *use healthy ASC from a patient - avoid immune rejection *many potential applications because they show “plasticity” (can be differentiated into other cells types) Complications with ASC Potential *normally in the body, the environment of stem cells limits their ability to specialise *experiments to date indicate that the cell’s environment determines what an adult stem cell will do *intensive research still in progress **found successful in some animals Induced Pluripotent Stem Cells *genes coding for specific proteins that control genetic switches were transferred to cells which triggered expression of other genes that created IPSC *although human IPS cells have been created, they are not yet able to be used within humans Complications with IPS *epigenetic differences **tips and centres of chromosomes are not reset to embryo-like stage **additional methylation *genomic abnormalities **incorporate extra cancer-causing genes and fewer tumour-suppressor genes Human Gene Therapy *treatment or prevention of disease by transfer of a functional gene *application is not limited to inherited disease but includes cancer and infectious diseases *current gene therapy practice involves gene addition, not correction or replacement *only somatic cells are treated **gene therapy on germ line cells is illegal *either the affected cells can be removed, the new gene added, and the cells returned to the body, or the new gene can be inserted directly *one possible method: Required Improvements in Gene Delivery *specificity and improvement of gene transfer *specificity, magnitude and duration of gene expression *avoiding stimulation of the immune system by some vehicles *manufacturing problems